recombinant dna part a

Preface DNA is the genetic material of virtually all living organisms. The physical mapping of genes, the sequence analysis of DNA, and the identi- fication of regulatory elements for DNA replication and transcription depend on the availability of pure specific DNA segments. The DNA of higher organisms is so complex that it is often impossible to isolate DNA molecules corresponding to a single gene in sufficient amounts for analy- sis at the molecular level. However, exciting new developments in re- combinant DNA research make possible the isolation and amplification of specific DNA segments from almost any organism. These new develop- ments have revolutionized our approaches in solving complex biological problems. Recombinant DNA technology also opens up new possibilities in medicine and industry. It allows the manipulation of genes from different organisms or genes made synthetically for the large-scale production of medically and agriculturally useful products. This volume includes a number of the specific methods employed in recombinant DNA research. Other related methods can be found in "Nucleic Acids," Volume 65, Part I, of this series. I wish to thank the numerous authors who have contributed to this volume, as well as the very capable staff of Academic Press, for their assistance and cooperation. I also wish to extend my appreciation to Stanley Cohen and Lawrence Grossman for their advice in planning the contents of this volume. RAY Wu xiii Contributors to Volume 68 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. DAVID ANDERSON (30), Genex Corporathm, Rockville, Maryland 20852 JAMES C. ALWINE (15), Laboratory of Molecular Virology, National Cancer In- stitute, National Institutes gf Health, Bethesda. Maryland 20014 S. L. AUCKERMAN (38), Department of Biology, The Johns Hopkins University, Baltimore, MaiTland 21218 KEITH BACKMAN (16), Department of Biology, Massachusetts Institute of Tech- nology. Cambridge, Massachusetts 02139 C. P. BAHL (7), Cetus Corporation, Berkeley, Califi)rnia 94710 RAMAMOORTHY BELAGAJE (8), Lilly Re- search Laboratories, Indianapolis, In- diana 46206 HANS-ULRICH BERNARD (35), Department o['Biology, University ~f California, San Diego, La Jolla, CaliJornia 92093 DALE BLANK (33), Rosenstiel Basic Medical Sciences Research Center and Depart- ment of Biology, Brandeis University, Waltham, Massachusetts 02154 FRANCISCO BOLIVAR (16), Deparamento Biologia Molecular, lnstituto de In- vestigaciones Biomedicas, Universidad Nacional Aatonoma de Mexico, Mexico 20, D.F., Mexico Apdo Postal 70228 ROLAND BROUSSEAU (6), Division of'Biolog- ical Sciences, National Research Council of Canada, Ottawa KIA OR6, Canada EUGENE L. BROWN (8), Synthex Research, Palo Alto, Califi~rnia 94304 DOUGLAS BRUTLAG (3), Department o[" BiochemisttT, Stanford University School qf Medicine, Stanford, Cal~)rnia 94305 JOHN CARBON (27, 31), Department of Bio- logical Sciences, University of Califi)rnia, Santa Barbara, Santa Barbara, California 93106 P. CHIU (29), Section of BiochemistiT, Molecular and Cell Biology, Cornell Uni- versity, Ithaca. New York 14853 LOUISE CLARKE (27, 31), Department t~f' Biological Sciences, University of Cali- fi)rnia, Santa Barbara, Santa Barbara, Califi)rnia 93106 STANLEY N. COHEN (32), Departments of Genetics and Medicine, StanJbrd Uni- versity School of Medicine, Stanford, Cal(~)rnia 94305 JO~N COLLINS (2), Gesellscht(fi flit Bio- technologische Forschung mbH. Masch- eroder Weg I. D-3300 Brutmschweig- St6ckheim, West Germany NICHOLAS R. COZZARELL1 (4), Departments of Biochemistry and Biophysics and Theoretical Biology, University of Chicago, Chicago, Illinois 60637 J. L. CULLETON (38), Department of Biof ogy, The Johns Hopkins University, Baltimore. Marylund 21218 R. P. DOTTIN (38), Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218 L. ENQU~ST (18), Laboratory of Molecular Virology, National Cancer Institute, National Institntes qf Health, Bethesda, Marykmd 20014 HENRY A. ERLI¢~ (32), Department of Medicine, Stanford Univel=sity School of Medicine, Stanfi~rd, California 94305 KAREN FAHRNER (33), Rosenstiel Basic Medical Sciences Research Center and Department ~[" Biology, Brandeis' Uni- t'ersity, Waltham, Massachusetts 02154 G. C. FAREED (24), Department of Micro- biology and hnmunology, Molecular Biol- ogy Institute, University of Cahi~rnia, Los Angeles, Los Angeles, California 90024 ix X CONTRIBUTORS TO VOLUME 68 DAVID FIGURSKI (17), Department of Micro- biology, College ~2f Physicians and Surgeons, Columbia University, New York, New York 10032 S. G. FISCHER (11), Department of Biologi- cal Sciences, State University of New York at Albany, Albany, New York 12222 B. R. FISHEL (38), Department of Biology, The Johns Hopkins University, Balti- more, Marvhmd 21218 MICHAEL L. GOLDBERG (14), Abteihmg Zelliologie, Biozentrium Der Universitiit Basel, CH-4056 Basel, Switzerhmd HOWARD M. GOODMAN (5), Howard Hughes Medical Institute Laboratory and the Department of Biochemistry and Bio- physics, University of Cahlfbrnia, San Fruncisco, Cal(fbrnia, 94143 MICHAEL GRUNSTEIN (25), Department qf Biology, University of California, Los Angeles, Los Angeles, Cali[brnia 90024 DONALD R. HELINSKI (17, 35), Department of Biology, University of Cahfornia, San Diego, La Jolla, California 92093 LYNNA HEREFORD (33), Rosenstiel Basic' Medical Sciences Research Center and Department qf Biology, Brandeis Uni- versity, Waltham, Massachusetts 02154 N. PATRICK HIGGINS (4), Department q[" Biochemisto', University qf Wyoming, Laramie, Wyoming RONALD H1TZEMAN (31), Department of Biological Sciences, University of Cali- fornia, Santa Barbara, Santa Barbara, California 93106 BARBARA HOHN (19), Friedrich Miescher Institut, CH-4002 Basel, Switzerland JANICE P. HOLLAND (28), Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032 MICHAEL J. HOLLAND (28), Department of Biochemistty, University of Connecticut Health Center, Farmington, Connecticut 06032 HANSEN M. HSIUNG (6), Division of Biologi- cal Sciences, National Research Council of Canada, Ottawa KIA OR6, Canada KIMBERLY A. JACKSON (28), Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032 MICHAEL KAHN (17), Department of Bac- teriology and Public Health, Washing- ton State University, Pullman, Washing- ton 99164 KATHLEEN M. KEGGINS (23), Department q[" Biological Sciences, University of Muo'lund, Baltimore County, Catons- ville, Mao, land 21228 DAVID J. KEMP (15), 1he Walker and Eliza Hall Institute of Medical Research, Post OJfice, Royal Melbowne Hospital, Vic- toria 3050, Australia H. GOBIND KHORANA (8), Departments oj" Biology and Chemisto', Massachusetts h~stitute of Technology, Cambridge, Massachusetts 02139 ROBERVO KOLTER (17), Department of Biology, University of" California, Sun Diego, La Jolla, Cal(fi)rnia 92093 L. F. LAU (7), Section qf Biochemistry, Molecular and Cell Biology, Cornell Unil,ersity, Ithaca, New York 14853 GAIL D. LAUER (34), The Biological Lab- oratories, Harvard University, Cam- bridge, Massachusetts 02138 LEONARD S. LERMAN (11), Department of Biological Sciences, State University of New York at Albany, Albany, New York 12222 RICHARD P. LIFTON (14), Department (2[ Biochemistry, StanJbrd University School q( Medicine, StanJbrd, California 94305 JOHN LIS (10), Section of Biochemistpy, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853 SHIRLEY LONGACRE (12), Parasitologie Ex- perimentale, Institut Pasteur, 75724 Paris, Cedex 15, France CONTRIBUTORS TO VOLUME 68 xi PAUL S. LOVETT (23), Department of Bio- k~gical Sciences, University of Maryland, Baltimore County, Catonsville, Maryland 21228 HUGH O. McDEVITT (32), Departments of Medicine and Medical Microbiology, Stanford University School c~f Medicine, Star,ford, California 94305 RAYMOND J. MACDONALD (5), Howard Hughes Medical Institute Laboratory and the Department of Biochemistry and Biophysics, University ~f California, San Francisco, San Francisco, Cahfornia 94143 BERNARD MACH (12), Department of Micro- biology, University of Geneva, CH 1205 Geneva, Switzerland R. E. MANROW (38), Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218 RICHARD MEYER (17), Department of Mi- crobiology, University of Texas, Austin, Texas 78712 D. A. MORRISON (21), Department of Bio- logical Sciences, University of Illinois, Chicago Circle, Chicago, Illinois 60680 JOHN f. MORROW (|), Department of Micro- biology, The Johns Hopkins School o[" Medicine, Baltimore, Mao, land 21205 S. A. NARANG (6, 7), Division of Biolog&al Sciences, National Research Council of. Canada, Ottawa KIA OR6, Canada TIMOTHY NELSON (3), Department of Bio- chemistry, Stanfi)rd University School of Medicine, Stanford, California 94305 BARBARA A. PARKER (15), Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305 BARRY POLlSKY (37), Department of Biol- ogy, Indiana University, Bloomington, Indiana 47401 A. F. PURCHIO (24), Department of Micro- biology and Immunology, Molecular Biology Institute, University of CaliJbrnia, Los Angeles, Los Angeles, Cal([brnia 90024 JOHN REEVE (36), Department of Micro- biology, Ohio State University, Columbus, Ohio 43210 JAKOB REISER (15), Department of Bio- chemistry, Stanford University School of Medicine, Stanford, California 94305 ERIC REMAUT (17), Laboratorium voor Moleculaire Biologie, Bijksuniversiteit Gent, B-9000 Gent, Belgium JAIME RENART (15), lnstituto de Enzi- mologia del C.S.1.C., Facultad de Medi- cina de la Universidad Autonoma, Arzobispo Morcillo s/n. Madrid-34, Spain ROBERT RICClARDI (33), Department of Bio- logical Chemistry, Harvard Medical School, Boston, Massachusetts 02115 RICrtARD J. ROBERTS (2), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 BRYAN ROBERTS (33), Rosentiel Basic Medical Sciences Research Center and Department ~f Biology, Brandeis Uni- versity, Waltham, Massachusetts 02154 THOMAS M. ROBERTS (34), Department of Biochemistry and Molecular Biology, Harvard University, Cumbridge, Mas- sachusetts 02138 MICHAEL ROSBASH (33), Rosenstiel Basic" Medical Sciences Research Center and Department of Biology, Brandeis Uni- versity, Waltham, Massachusetts 02154 R. J. ROTHSTEIN (7), Department of Micro- biology, New Jersey School of Medicine, Newark. New Jersey 07103 STEPHANIE RUBY (33), Department of Bio- logical Chemistry, Harvard Medical School. Boston, Massachusetts 02115 MICHAEL J. RYAN (8), Microbiological Sciences, Schering Corporation. Bloomfield, New Jersey 07003 A. SEN (13), Meloy Laboratories Inc., Springfield, Virginia 22151 xii CONTRIBUTORS TO VOLUME 68 LUCILLE SHAPIRO (30), Department t~f'Mo- lecular Biology, Albert Einstein College of Medicine, Bronx, New York H. MlCrtAEL SHEPARD (37), Department ~[" Biology, bldiana University, Blooming- ton, hldiana 47401 F. SHERMAN (29), Department qfRadiation Biology and Biophysics, University of Rochester, School oJ' Medicine, Roches- ter, New York 14642 M. SHOVAI3 (13), Laboratory of Viral Car- cinogenesis, National Cancer Institute, National Institutes ~)1 Health, Bethesda, Marylund 20014 A. M. SKALKA (30), Department ¢~f Cell Biology, Roche Institute ~[" Malecular Biology, Nutley, New Jersey 071IO EDWIN SOUTHERN (9), M.R.C. Mammalian Genome Unit, King's Building, Edin- burgh EH9 3JT, Scotland GEORGE R. STARK (14, 15), Department ~f" Biochemistry, Stanford University School ~]" Medicine, Stat~jbrd, Cal(fornia 94305 N. STERNBERG (|8), Cancer Biology Pro- gram, Frederick Cancer Research Cen- ter, Frederick, Maryland 21701 J. I. STILES (29), Department of Radiation Biology and Biophysics, University of Rochester, School ~f Medicine, Rochester, New Yark 14642 M. SUZUKI (22), Boyce Thompson Institute, Cornell University. Ithaca. New York 14853 A. A. SZALAY (22), Boyee Thompson Insti- tute, Cornell University, Ithaca, New York 14853 J. W. SZOSTAK (29), Sidney Faber Cancer Institute, Boston, Massachusetts 02115 CHRISTOPHER THOMAS (17), Department of Biology, University ~)f" Cul([brnia, San Diego, La Jolla, Cal(fornia 92093 B K. TVE (29), Section of Biochemisto', Molecuhtr and Cell Biology, Cornell Universio,. Ithaca, New Yor,~" 14853 GEOFFRE'¢ M. WAHL (15), Department qf Biochemisto', Stanford University School qf Medicine, Stanford, CaliJbrnia 94305 JOHN WALLIS (25), Department of Micro- biology and Immunology, Molecular Biol- ogy Institute. University ~1" CaliJornia, Los An,~,eles, Los Angeles, California 9OO24 JEEFREY G. WIkLIAMS (14), Imperial Cancer Research Fired, Mill Hill, London NWT, England SAVIO L. C. Woo (26), Howard Hughes Medical Institute Laboratory and De- partrnent of'Cell Biology, Baylor College ~0 r Medicine, Texas Medical Center, Houston. Texas 77030 JOHN WOOLFORD (33), Rosenstiel Basic Medical Sciences Research Center and Department ~( Biology, Brandeis Univer- sit),, Waltham. Massachusetts 02154 RAy Wu (7, 10, 29), Section of Biochemistry, Maleeular and Cell Biology, Cornell Uni- versity, Ithaca, New York 14853 ROBERT C A. YANG (10), Section of Bio- chemisttT, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853 [1] RECOMBINANT DNA TECHNIQUES 3 [1] Recombinant DNA Techniques By JOHN F. MORROW The recombinant DNA method consists of joining DNA molecules in vitro and introducing them into living cells where they replicate. Research using this method is relatively new and fast-moving. In only 6 years of re- combinant DNA research, a number of significant accomplishments have been made. Two mammalian hormones have been produced in bacteria by means of synthetic DNA. m Polypeptides similar or identical to several found in eukaryotes have been synthesized in Escherichia coli. 3-~° These achievements promise a new, inexpensive means of large-scale produc- tion of selected peptides or proteins. Furthermore, using recombinant DNA, somatic recombination of immunoglobulin genes has been estab- lished, 11 and a large number of variable-region genes have been found. TM Intervening sequences (introns) have been found in the DNA of eu- karyotic cells. 1a-t6 I would like to mention the origins of this versatile new technology be- fore describing recent advances. The isolation of mutant E. coli strains unable to restrict foreign DNA (cleave it specifically and degrade it) laid i K. Itakura, T. Hirose, R. Crea, A. D. Riggs, H. L. Heyneker, F, Bolivar, and H. W. Boyer, Science 198, 1056 (1977). 2 D. V. Goeddel, D. G. Kleid, F. Bolivar, H. L. Heyneker, D. G. Yansura, R. Crea, T. Hirose, A. Kraszewski, K. Itakura, and A. D. Riggs, Proc. Natl. Acad. Sci. U.S.A. 76, 106 (1979). K. Struhl, J. R. Cameron, and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 73, 1471 (1976). 4 B. Ratzkin and J. Carbon, Proc. Natl. Acad. Sci. U.S.A. 74, 487 (1977). D. Vapnek, J. A. Hautala, J. W. Jacobson, N. H. Giles, and S. R. Kushner, Proc. Natl. Acad. Sci. U.S.A. 74, 3508 (1977). e R. C. Dickson and J. S. Markin, Cell 15, 123 (1978). L. Villa-Komaroff, A. Efstratiadis, S. Broome, P. Lomedico, R. Tizard, S. P. Naber, W. L. Chick, and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 75, 3727 (1978). s A. C. Y. Chang, J. H. Nunberg, R. J. Kaufman, H. A. Erlich, R. T. Schimke, and S. N. Cohen, Nature (London) 275, 617 (1978). 90. Mercereau-Puijalon, A. Royal, B. Carol, A. Garapin, A. Krust, F. Gannon, and P. Kourilsky, Nature (London) 275, 505 (1978). 1o T. H. Fraser and B. J. Bruce, Proc. Natl. Acad. Sci. U.S.A. 75, 5936 (1978). 11 C. Brack, M. Hirama, R. Lenhard-Schuller, and S. Tonegawa, Cell 15, 1 (1978). n j. G. Seidman, A. Leder, M. Nau, B. Norman, and P. Leder, Science 202, 11 (1978). 13 D. M. Glover and D. S. Hogness, Cell 10, 167 (1977). x4 R. L. White and D. S. Hogness, Cell 10, 177 (1977). 15 p. K. Wellauer and I. B. Dawid, Cell 10, 193 (1977). 1, S. M. Tilghman, D. C. Tiemeier, J. G. Seidman, B. M. Peterlin, M. Sullivan, J. V. Maizel, and P. Leder, Proc. Natl. Acad. Sci. U.S.A. 75, 725 (1978). METHODS IN ENZYMOJ.OGY, VOL. 68 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN O- 12-181968-X 4 INTRODUCTION [1] part of the foundation. 17 The discovery of site-specific restriction endonu- cleases lsa9 also contributed (see Nathans and Smith, ~° Roberts, 21 and this volume [2], for review). Two general methods for joining DNA molecules from different sources were found. 2z-24 Particularly useful was the first enzyme found to create self-complementary, cohesive termini on DNA molecules by specific cleavage at staggered sites in the two DNA strands, the EcoRI restriction endonuclease. 25-2r It was used in the first in vitro construction of recombinant molecules that subsequently replicated in vivo. 2s What can be done by the recombinant DNA method? Principally three sorts of things: 1. Isolation of a desired sequence from a complex mixture of DNA molecules, such as a eukaryotic genome, and replication of it to provide milligram quantities for biochemical study. 2. Alteration of a DNA molecule. One can insert restriction endonu- clease recognition sites, or other DNA segments, at random or predeter- mined locations. One can also delete restriction sites, or DNA segments between such sites, by techniques that permit joining any two DNA ter- mini after their appropriate modification. Such an alteration can be helpful in determining the functions performed by various parts of a DNA se- quence. This is attractive where efficient means of fine-structure genetic analysis of random mutations are lacking, as in animals and plants. 3. Synthesis in bacteria of large amounts of peptides or proteins that are of interest to science, medicine, or commerce. Before indicating specifically the most useful methods for obtaining each of the above goals, we look at recent advances in the basic tech- niques. The essential ingredients of a recombinant DNA experiment are: 17 W. B. Wood, J. Mol. Biol. 16, 118 (1966). is H. O. Smith and K. W. Wilcox, J. Mol. Biol. 51, 379 (1970). ~9 T. J. Kelly, Jr. and H. O. Smith, J. Mol. Biol. 51, 393 (1970). 2o D. Nathans and H. O. Smith, Annu. Rev. Biochem. 44, 273 (1975). 21 R. J. Roberts, Gene 4, 183 (1978). 22 p. E. Lobban and A. D. Kaiser, J. Mol. Biol. 78, 453 (1973). 23 D. A. Jackson, R. H. Symons, and P. Berg, Proc. Natl. Acad. Sci. U.S.A. 69, 2904 (1972). 24 V. Sgaramella, J. H. van de Sande, and H. G. Khorana, Proc. Natl. Acad. Sci. U.S.A. 67, 1468 (1970). 25 j. E. Mertz and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 69, 3370 (1972). 2e j. Hedgpeth, H. M. Goodman, and H. W. Boyer, Proc. Natl. Acad. Sci. U.S.A. 69, 3448 (1972). z7 V. Sgaramella, Proc. Natl. Acad. Sci. U.S.A. 69, 3389 (1972). 2s S. N. Cohen, A. C. Y. Chang, H. W. Boyer, and R. B. Helling, Proc. Natl. Acad. Sci. U.S.A. 70, 3240 (1973). [1] RECOMBINANT DNA TECHNIQUES 5 1. A DNA vehicle (vector, replicon) which can replicate in living cells after foreign DNA is inserted into it. 2. A DNA molecule to be replicated (passenger), or a collection of them. 3. A method of joining the passenger to the vehicle. 4. A means of introducing the joined DNA molecule into a host orga- nism in which it can replicate (DNA transformation or transfection). 5. A means of screening or genetic selection for those cells that have replicated the desired recombinant molecule. This is necessary since transformation and transfection methods are inefficient, so that most members of the host cell population have no recombinant DNA repli- cating in them. This selection or screening for desired recombinants pro- vides a route to recovery of the recombinant DNA of interest in pure form. Since a thorough review of recombinant DNA was completed in 1976, 29 I will concentrate on progress since then. Cloning Vehicles Plasmids Many bacterial plasmids have been used as cloning vehicles. Cur- rently, E. coli and its plasmids constitute the most versatile type of host-vector system for DNA cloning. A number of derivatives of natural plasmids have been developed for cloning. Most of these new plasmid vehicles were made by combining DNA segments, and desirable qualities, of older vehicles (Table I). All those listed have a "relaxed" mode of replication, such that plasmid DNA accumulates to make up about one-third of the total cellular DNA when protein synthesis is inhibited by chloramphenicol or spectino- mycin. ~0 pBR322 is now the most widely used plasmid for cloning of DNA. One of its virtues is that it has six different types of restriction cleavage termini at which foreign DNA can be inserted. A very detailed restriction enzyme cleavage map and DNA sequence information are also important. 31a2 The PstI site in the Ap (penicillinase) gene has further advantages. If dG ho- mopolymer tails are added to Pst-cleaved pBR322 DNA, and dC homo- polymer tails to the DNA to be inserted, the PstI sites are reconstituted in 29 R. L. Sinsheimer, Annu. Rev. Biochem. 46, 415 (1977). 3o A. C. Y. Chang and S. N. Cohen, J. Bacteriol. 134, 1141 (1978). at j. G. Sutcliffe, Proc. Natl. Acad. Sci. U.S.A. 75, 3737 (1978). 32 j. G. Sutcliffe, Nucleic Acids Res. 5, 2721 (1978). 6 INTRODUCTION [ 1] ~ Z ~ z u.l Z O'r' ~.~ • 7. E < o o O e~ ,4 o o < E N "O r~ "4 o < < 0o .< .< E ~v III i~. r r [] .4 e~ O0 e~ E O 'r" o e~ ¢N = [] e~ [1] RECOMBINANT DNA TECHNIQUES 7 ~ ~ ,~ ~ ~ "- ,,., .~ ~ ~, _o~ ~ ~ ~ ~ ~ ,~.~_ ~ ~ ~ ~ .~ ~ .< < ,,o I I I .< ,< < © e4 i D'- 0 • ~, , "2 o 2 r- .I~ .~ ,~ O0 • ~I" ~. 0 ~ ~ ~ :~ ~ ~.~ ~'~ ~. ~. ~ ,-~ .~ = " r- ~ "~ • 0 0 P", • ~ >,~,~ ~ ~ • ~ .~ o',, . ~ ~ ~ ~ g ~-~ ~. ~ ~ .~ ~ -~ ~- ,, ~ .~o ~'~ ~ .'~ ~. ~:~ ~_~ ~-o =~ ~ ,~.~ ~,~.,~:~ . ~ . "~,~.o.~ .o. . Ed~uu ~ u [...]... ¢-q ¢,q g >, ca A A A -2 AA AA ~ss AA A ~ A A -~ A 2sss Z ~v.~ ~ z ZDe e.e.e.e, ~ 0 o Q 0 M., ~ t,4 t~/ ~ =~ t~ ~ ° O 0 0 ° O O ~, 1,4 k 32 A A A AA AA ~AAAAAA AAAAA AAA A A AA AAA A A AA 33 P~ o o "d A z A AA ~ ~ N ~ - A A A A A O~.~.~ z~ ;34 A A ~.~.O O O ~ 0o exl AA AA A AA t~ A A u~ ~o z z~z~-~ ZzZzg ,,~ z z z z z z ~ ~ ~ ~ ZZ'~L ;= =_ < ~Z ~ ~ ,, ~ o ~ ~ , ~• ~ ~ > -"~ o ~ ~ ~'N ~ ~ • '~, o... experiments using EcoRIcleaved pBR325 DNA 37 The pJC plasmids in Table I are representatives of a class of plasmids called cosmids because they have the h phage cos DNA site required for packaging into h phage particles, and they replicate as plasmids because of their ColE1 DNA segments Their chief advantage is the fact that pJC DNA ligated to foreign DNA can be packaged in vitro and used to infect E co/i... approach the maximum possible capacity for inserted DNA in a nondefective ~, phage vector (Table II) Charons 4 and 8-11 are believed to be able to replicate more than 22 kb of inserted DNA A number of Charon phages contain a l a c Z (fl-galactosidase) gene Substitution of foreign DNA in place of the l a c P O Z DNA segment makes the phage lacO- Growth on a lac + bacterial strain on plates containing a chromogenic... Sobel, T Yamamoto, S L Adams, R DiLauro, V E Avvedimento, B deCrombrugghe, and I Pastan, Proc Natl Acad Sci U.S .A 75, 5846 (1978) s9 A Efstratiadis, F C Kafatos, and T Maniatis, Cell 10, 571 (1977) go H C Heindell, A Liu, G V Paddock, G M Studnicka, and W A Salser, Cell 15, 43 (1978) [1] RECOMBINANT DNA TECHNIQUES 21 bumin cDNA plasmid also indicates faithful cloning of the mRNA's sequence, a~ The faithfully... so that single-stranded termini are produced 2°, 2a These can be annealed with D N A from another source and ligated to form recombinant DNA. ~S,39 The cloning vehicle D N A can also recircularize by itself, with no inserted DNA As a result, 7 5 - 9 0 % of the transformants usually contain vehicle alone instead of recombinant DNA 3ha9 A method to pr.event resealing of the vehicle D N A is removal of... plasmid DNA transforms bacteria with the same high efficiency as supercoiled plasmid DNA, it can give rise to clones containing vehicle plasmid rather than a recombinant Inactivation of a genetic marker is not an infallible indication of DNA insertion For instance, when pBR324 is digested by Barn HI, Hin dIII, or SalI restriction endonuclease, ligated with other similarly digested DNA, and used to transform... to add a homopolymer extension, e.g., polydeoxyadenylate, to each 3'-end of the vehicle DNA, and a complementary extension to each 3'-end of the passenger DNA (see this volume [3]) An attractive application is to add dG tails to PstI-cleaved pBR322 DNA and dC tails to the DNA to be inserted, anneal, and transform E coli with the DNA P s t I sites are reconstituted on both sides of the inserted DNA in... incubation with terminal transferase 22" 2a The exonuclease is not necessary TM However, a technical precaution which is useful in cloning large eukaryotic DNA fragments is to eliminate small polynucleotides ( . Wyoming, Laramie, Wyoming RONALD H1TZEMAN (31), Department of Biological Sciences, University of Cali- fornia, Santa Barbara, Santa Barbara, California 93106 BARBARA HOHN (19), Friedrich Miescher. circular plasmid DNA transforms bacteria with the same high efficiency as supercoiled plasmid DNA, it can give rise to clones containing vehicle plasmid rather than a recombinant. Inactivation. phage cos DNA site required for packaging into h phage particles, and they replicate as plasmids because of their ColE1 DNA segments. Their chief advantage is the fact that pJC DNA ligated to